Microbial tests from chronic wounds

If we suspect that we have an infection in the wound and consider starting a course with antibiotic treatment we should  try to find out which bacteria might be causing the infection.  Apart from advanced techniques like PCR analysis which is only found at very few institutions in Africa  we are left with two choices.  We can either do a bacterial swab and transfer the swab onto a culture plate to multiply the bacteria in the course of a few days.  Once the bacteria have multiplied they are transferred to a glass plate, stained ( usually with Gram stain) and examined under a microscope.  The other option is to take a sample from the wound and transfer this directly to a glass plate and do a Gram stain prior to examination under the microscope.  The advantage with the latter method is that it is cheap and  quick - within about 20 minutes you may already have examined the sample. The disadvantages are that if there are few bacteria you may miss them. Also, a direct examination will carry along cell debris and other artefacts which can make visualization of the bacteria more difficult. Also, you will not be able to do an analysis of bacterial resistance.  When using the culture plate technique we are able to place small bits of antibiotica laden paper onto the culture to check which antibiotics have best effect on that specific strain of bacteria. 

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Figure 1. In general there are two methods to check which bacteria may be causing an infection in the wound.  The first method ( A) is by transferring the swab to a growth medium ( often blood agar) to multiply the bacteria first.  In this way we get a higher concentraion of bacteria which makes identification of the strain much easier.  The downside is that the culture process usually takes 2-3 days. After the bacteria have been cultured a regular stain and examination under the microscope is done.

The direct method (B) involves taking a sample from the woundbed and transferring this directly to a glass slide, staining it and examining the slide immediately.  because the bacterial numbers are lower sometimes they can be harder to identify. 

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Figure 2  Another advantage when culturing bacteria on a growth medium is that we can test how different antibiotics perform on that particular strain. For this purpose small round paper plates saturated with different antibiotics are laid on the culture and then we observe how close to these antibiotics the bacteria grow. In the image above we see that the bacteria are completly resistant to the antibiotic witht he blue arrow. Here we see bacteria growing right up to the paper contianing that particular antibiotic.  This is very useful information when choosing an antibiotic for treating the patient. 

When should you do a bacterial swab from a chronic wound?

 

There are far too many bacterial swabs taken from chronic wounds!  This has led to an overuse of antibiotics in chronic wounds worldwide.  It is very important to understand that you ALWAYS will  get some sort of bacteria growing from a chronic wound.  It is for example very usual to find staphylococcus aureus in many chronic ulcers. It is also not unusual to find pseudomonas aeroginosa, e-coli or other enterococci in chronic wounds.  This does not mean that the patient needs antibiotics!  It is completly wrong to think. " let`s do a bacterial swab and see what turns up - if we find pathological bacteria we will start with a course of antibiotics". 

Figure 1 Rememeber that you always will find bacteria in a chronic wound in the same way that you always will find bacteria living on our skin.  If you take a routine swab from our skin you will most likely get a nice mixture of potentially pathological bacteria although you are perfectly healthy.  You would not place a person on a course of antibiotics only because you found staphylococcus aureus in a swab from the skin, right?

As a rule of thumb: if you are thinking of starting a patient with a chronic ulcer on a course of antibiotics you should do a bacterial swab. Once the swab has been performed you can start the antibiotic treatment. As soon as the test results are back you have to check if the antibiotics you have adminstered are the right type for the bacterial strains shown. If you haev a good laboratory at hand they will have done a resistancy test to show which antibiotics are most advisable in this specific case. 

 

The question, however, is - should you start the patient on antibiotics at all? If there are clear signs of a significant infection or the patient even has systemic signs of infection then antibiotic treatement is most likely necessary.  Just having some redness around a chronic ulcer is by no means a sure sign of infection - in most cases this is simply the bodies inflammatory response to the fact that there is a hole in the skin at this place. 

At the tissue viability centre where we work we very rarely use antibiotics even though we quite often are dealing with large and complicated ulcers.  We never never do a routine bacterial swab of an ulcer just to be curious about who is living in the bottom of the ulcer. 

In diabetic patients with deep foot ulcers however, we have a low threshold for starting with antibiotic treatment, especially if the ulcer is close to bone- or tendon structures.  This is because these ulcers quickly can deteriorate and we do not always see the typical infection signs of redness, swelling and pain in diabetic patients.  In other words- in  more challenging diabetic feet ulcers we  routinely take bacterial swabs because we often use antibiotics here. 

Another situation where we might take a bacterial swab although the wound is not clearly infected is when we have an ulcer that is not healing as expected. In such cases you should also take a tissue biopsy for a histological analysis but you may also consider a bacterial swab at the same time.  Sometimes colonization with for example streptococci or pseudomonas can be invisible to the eye but impair healing to such an extent that the wound just stalls. This doesnt mean that the presence of streptococci or pseudomonas in a wound always means trouble - but in some cases it does.  If you believe that the problem with the wound is an  unfortunate colonization with a strain of bacteria you should do a swab and first try a topical antibacterial dressing to try to eleminate the bacteria in this way.  There is a wide choice of antimicrobial dressings available - ranging from silver coated dressings and iodine dressings to honey- and sugar preparations. 

How should you do a microbial swab?

Our standard swab for bacterial specimen collection and transportation is a black capped swab containing Amies gel medium with charcoal. It is intended for recovery of aerobic, anaerobic and fastidious bacteria. The charcoal in the medium neutralizes substances which are toxic to sensitive bacteria. All swabs used should be CE marked and meet the Clinical Laboratory Standard Institute approved standard M40-A2. Obviously if you only can get hold of non- CE marked swabs then you have to use these and hope that they meet basic standards. 

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Figure 2  Amies medium is the standard swab for bacterial cultures from chronic ulcers. Remember that the swab is sterile and that is important not to contaminate the swab while you take the sample. In other words, it is important that you only come into contact with the desired area in the wound and are careful not to touch the periwound area, the patients clothes or your gloved fingers with the swab tip. 

A chronic ulcer usually contains a mixture of different bacteria.  When we are dealing with an infection of the wound only rarely are all of these bacteria involved in the infection. In most cases only one of the bacterail strains is the real culprit. These are usually the bacteria that are left behind when we have rinsed the wound thoroughly.  So - to find the real culprits we have to wash away all the unimportant bystanders and expose those who are remaining.  To do this we usually have to debride the wound well and then rinse it thoroughly.  At our courses we are often asked whether this wont wash away all the bacteria.  Don`t worry - you will never manage to wash away all the bacteria and you can be quite sure that the relevant bacteria still will be hiding in the surface of the wound bed ready to be detected with your swab.  The more you rinse the woundbed the more exact your swab will be! 

Only use sterile saline when rinsing the wound brior to taking a swab. Ideally the rinsing solution should be around body temperature.  This is because cold rinsing solutions may make the bacteria less vital. Besides, the wound itself prefers not to be cooled down.  Do not use antimicrobial rinsing solutions like polyhexanide (prontosan), superooxidized water or vinegar based rinses as these may give you a false negative bacterial swab! 

When you take the swab press or rub the tip of the swab gently over the deepest portion of the wound bed - this is usually the best catchment area. If you have areas of exposed bone or tendon take the bacterial samples from these tissues. If the surface is dry then moistening the tip of the swab with sterile saline will help bacteria to adhere to the swab. Sample the entire surface of the area suspected to be infected using a zig-zag motion whilst simultaneously rotating the swab between the fingers.  Place the swab into the transport medium immediately after swabbing. Be very careful not to contaminate the tip of the swab as you retreat it out of the wound.  

Filling ot the microbiology request for the lab

Remember to write the patients name on the outside of the swab cover and on the request form to the lab!  This should be unnecessary to mention but is surpsingly often forgotten. A bacterial swab without a name is basically useless and is usually discarded. 

The microbiologist is dependent on a lot of clinical information to be able to make a sensible decision about which of the bacteria in the culture are most relevant.  The microbiologist wil find it useful to know which kind of ulcer the patient has. In a venous leg ulcer for example usually only one bacteria is the cause of the infection. In a diabetic foot ulcer more often two ( or more) bacteria may be actively involved in the infection The labortaory also needs information about where the ulcer is located - do not just write " pressure ulcer" on the request. If you write "sacral presure ulcer" the microbiologist will understand why there are lots of e-coli and enterocoocus bacteria in the swab - after all the sacrum is in close vicinity to the anus and it is common to find intestinal bacteria in sacral pressure ulcers.  You have to write whether the patient has been using antibiotics recently and whethetr antimicrobial rinsing solutions or antimicrobial dressings ( silver, idodine, honey, sugar?) have been used.  It is also really important to write that your swab has been taken from a cleaned ( and debrided) woundbed. 

Storing the swab

A bacterial swab is a fresh product and should be sent to the lab as soon as possible.  If you because of logistical reasons cannot send it the same day you should store the swab in a fridge at a temperature between 4- 8 ° C til the enxt day.  Be aware though that the sample may not be as good the next day - you may not get growth of all bacteria.  The best is to send the swab to the lab the very same day! 

 

If you are working off the grid, with no access to a laboratory what can you do?

In most areas in Africa, even in quite remote areas there is usually a clinic or dispensary where they may be someone with a microscope. Whether they are capable of doing a basic bacterial culture analysis is another question.  Often these health workers may have some training in analyzing blood slides to test for malaria for example which only requires basic staining techniques.  Doing a bacterial culture with culture plates which have to be incubated etc. is another thing.  However, you will find that most often - if they are able to do a malaria stain then they also will be able to do a Gram stain for bacterial analysis.

Remember that you do not necessarily have to culture the bacteria before analyzing them. You can take a swab directly from the wound and smear the swab directly onto a glass plate and stain this with a gram stain. The problem with this " direct" technique is that you will see a lot of artefacts ( cell debris etc) and only a few bacteria as they have not been concentrated by culture on a agar plate.  With a little training however you will be able to recognize at least the most common pathogenic bacteria.

The Gram stain provides preliminary results on whether bacteria are present ( they will always be present in a chronic wound!)  and the general type, such as the shape and whether they are Gram-positive or Gram-negative. This will give a better indication of which antibiotic to use rather than just taking an educated guess by just looking at the infected wound.

If you are using this direct method you can use a clean cotton q-tip to transfer the bacteria from the wound bed onto the glass slide.  Clean and rinse the wound thoroughly as you would do when doing a swab before taking your sample. 

Again, if you have access to a modern microbiological laboratory who can culture the swab to concentrate the bacteria and even do antibiotic resistance tests then this is great.  If you do not have access to this type of service you will have to manage with doing a direct gram stain. 

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Figure X With some basic training it is not diffucult to do a gram stain and to at least identify the most common pathogenic bacteria.  The disadvantage of this technique as opposed to culturing the bacteria first is that there often are quite a lot of artefacts in the slide and the number of bacteria is often quite low making the interpretation more difficult. 

Video 1 A short presentation on how to do a gram stain.  You will need a heat source, three types of dyes and alcohol- all these items are very cheap and  usually available from various suppliers in Africa. The staining technique is very easy to learn - the interpretation of the slides under the microscope requires some more training. 

Hvow do you interpret the microbiological results?

If you are working at a clinic where you regularly send bacterial swabs to a laboratory you have to have a system for checking the results as soon as they return and checking which antibiotic treatment the patient has received.  It should seem obvious that this is what should be done - but from experiences at our own clinic we regularly see that someone  forgets to check whether the antibiotic prescribed really is adequate for the bacteria found in the culture.  For example, if we prescribed a beta-lactam antibiotic to a patient with a wound infection and the bacterial swabs shows primarily growth of enterococci then we should switch to a different antibiotic. You have to establish a routine for this and determine who is responsible for phoning the patient informing him/her about the need to switch antibiotics. If you have a laboratory which performs antibiotic resistance tests then you can tailor the antibiotic treatment even more exact. 

Even if we have cleaned and rinsed the wound thoroughly before taking the sample we may end up with a micture of bacteria in the lab report.  This will make the choice of antibiotics more difficult. Remember- it is quite rare to have several bacteria that are equally involved in causing an infection. Usually only one strain of bacteria found in the culture is responsible for the infection.  Instead of prescribing broad spectrum antibiotics we should use our clinical experience to make an educated guess about who is the true culprit and use narrow spectrum antibiotics as often as possible.  The smell of the infected wound can give us important clues - does the wound smell rotten or even faeces like - then most often some intestinal bacteria is the culprit of the infection.  Staphylococcal infections do not usually make smelly wounds.  If the skin around wound is intense red and the redness is spreading quite rapidly then you should suspect streptococci or staphylococci to be the culprits.  If there is greenish exudate in the wound this is indicative of an infection with pseudomonas. 

If you have debrided the wound well and rinsed it thoroughly you will most often get a much more exact lab result - if you are lucky the results will only show one strain of bacteria and the choice of antibiotic is much easier. 

Figur 3  Results from a bacterial swab from a chronic wound that was cleaned well and rinsed thoroughly prior to taking the swab. The results show only staphylococcus aureus growing in the culture and the antibiotic resistance testing showed resistancy to erythromycon and clindamycin.  Note: the lab report is in the norwegian language but the essential part of the text is universally understandable.   The letters "S" And "R" behind each antibiotic stand for sensitive and resistant respectively. 

Figur 4  An example of a poorly performed swab from a chronic wound.  The wound was not cleaned rinsed properly before the sample was taken and the test results  will give you a headache.  The microbiologists found 6 different strains of bacteria growing in the culture - each one of the strains was equally abundant!  We can ssure you that not all six strains were responsible for the patients infection in the wound.  And trying to interpret the antibiotic resistancy test is surely a challenge - which antibiotic should we use here???  This bacterial swab was a waste of time and we should definitely not prescribe three different antibiotics to cover all six strains of bacteria!  We hope that this example clearly demonstrates the problems you will face if you do not clean and rinse the wound thorughly before douing a swab! Note: the lab report is in the norwegian language but the essential part of the text is universally understandable.   The letters "S" And "R" behind each antibiotic stand for sensitive and resistant respectively. 

How do you take a bacterial swab from bone where you suspect osteomyelitis?

Osteomyelitis is an infection of the bone. The treatment necessitates many weeks, sometimes months of antibiotic treatment.  It is therefore crucial that we are as certain as can be that we have chosen the right antibiotic for the bacteria that are causing the bone infection.  It is simply quite tragic if you treat the patient for 12 weeks with an antiobiotic only to discover after that time that the treatment is not effective because you chose the wrong antibiotic. 

 

If you are treating an osteomyelitis it is therefore essential to have secured a bacterial analysis before starting the antibiotic treatment.   If a patient already has been taking some antibiotics without an swab having been taking you should pause the antibiotic treatment for 10-14 days and then do the bacterial swab.

Even if you have an open ulcer above the infected bone - it is not very helpful to just take a bacterial swab from the wound - this will not necessarily tell you which bacteria is living in the bone.  Essentialy you have to take a bacterial swab from within the bone.

It is not a good idea to go through the open wound to take the swab from the bone as you almost always will touch the woundbed with the swab as you enter the bone - the swab will get contaminated with maybe completly irrelevant bacteria. Sometimes however,  there is a large visible bone defect in the open wound and it may be possible to enter the bone with the swab without touching the wound bed.  

The gold standard method is to enter the bone through a seperate incision in an area which is not affected by the wound, use a surgical tool to open the bone and insert the swab there. This will give you the best test results without any danger of contamination of the wound. This obviosuly demands training and resources which are not availabel everywhere.  If there is a deep osteomyelitis of the pelvis sometimes CT- or MRI guided techniques are used at high level medical centres to take precise bacterial tests from deeper within the body. 

Even though we have the described gold standard above, many wound practitioners settle for taking a bacterial swab through the open wound because this is more practical and easy to perform.  If you clean the wound very thoroughly and rinse it very well beforehand - in this situation you could even use antibacterial rise solutions like super oxidized water or 3,5% vinegar - and a very careful not to touch the wound bed with the swab you can get a fairly accurate sample.  At our clinic we often use this technique.

If the surface of the bone is necrotic you can be very sure that there is an osteomyelitis lurking beneath and you can use a ring curette to remove the upper millimeters of the necrotic bone and take a swab from beneath here. 

Cultures from bone swabs can be disapointing and a tip to get more consistent results is to scrape away some of the rotten bone - place this in a sterile syringe closed with a luer lock. Fill the syringe with about 6-8 ml of sterile saline and after replacing the plunger shake the syringe vigorously for  about a minute to wash the bacteria out of the bone.  The resulting broth is injected equally into blood culture mediums ( anaerobic and aerobic) and sent for culturing to a laboratory.  With this method we have more often succeeded in isolating the bacteria causing the osteomyelitis. You will obviosuly need blood culture medium avialable for this and preferably a laboratory that can do the analysis. if you do not have such a laboratory then you can place the blood culture mediums in a cabinet at 37 degrees centigrade and let the bacteria multiply for 12-24 hours.  After the first 12 hours you can  aspirate a few millitres of each glass and do a gram stain from each glass.  If you do not see enough bacteria to make a definite diagnosis wait for another twelve hours and repeat the stains. 

If you have exposed bone in the wound but the bone is white and healthy looking you must think twice about creating a defect in the bone to take a bacterial sample!!! Don`t do it is what we are saying!  If you have made a hole in the bone bacteria from the wound will definitely get into the bone and cause an osteomyelitis.  You should only open the bone when you are 100% sure that there is an osteomyelitis lurking beneath - that is either you can clearly see necrotic bone at the bottom of the wound with your eyes or you have the diagnosis confirmed by an x-ray, CT scan or even MRI-scan.  

Figur 5  The image above shows a bone biopsy probe being inserted into the distal phalang of a toe under CT scan guidance. You can clearly see changes in the bone indicative of osteomyelitis (messy appearnce of the bone structure).The ulcer was on the plantar side of the toe and the probe is passed into the bone at an area with intact skin as not to contaminate the probe with irrelevant bacteria from the wound. This is the gold standard for taking a microbial biopsy from an osteomyelitis site.  The procedure can also be done using regular x-ray or fluoroscopy of that is available.  In easily acessed areas like the toes the procedure can also be done without radiologic imaging support. 

Figur 6  We prefer blood culture mediums to culture bacteria when we take microbial samples from bone. As we described above this involves scraping some necrotic bone from the site of the osteomyelitis, placing this in a sterile container with about 6-8 ml of sterile saline and shaking this well for about a minute to wash bacteria out of the bone.The resulting broth is injected into the culture mediums and incubated for 12-14 hours, sometimes longer. After this we do gram stains from the mediums or get the cultures analyzed in a laboratory. This method has given us consistent,  reliable results from osteomyelitis samples where the bacterial counts may be lower than in other infected tissue. 

What about checking for fungal infections?

In woundcare fungal infections are often overseen.  We are usually much more concerned about bacterial infections.  And indeed, without a doubt are bacterial infections what we encounter mostly.

 

Most commonly we encounter fungal infections in the toe nails of our patients and often we ignore this as the ulcers  on the legs and feet demand our attention more.  Note that sometimes a infected toenail can give rise to an infection of the nailbed and this is often overseen as the discouloured toenail hides from what is underneath.  This sort of situation is not rare in diabetic patients.

Another common site for fungal infections is the moist area between toes.  Whilst this condition, commonly known as athletes foot ,can cause itching and mild discomfort in healthy individuals it can cause limb threatening secondary infections in diabetic patients and those with reduced immune system. 

Another quite common situation are fungal infections arising in moist skin folds, for example under the breasts , in the groin areas or in abdominal folds in overweight patients.  These fungal infections can be quite aggressive causing large ulcerations and secondary infections. 

In Africa where we more often encounter HIV patients fungal infectiosn of the skin or even systemic fungal infections are something which we always must keep in mind.

Be aware however that opportunistic fungi also can colonize a chronic wound and get a wound to stall without making any big fuss out of it- in other words there are no if only few tell-tale signs of a fungal infection.  We recall a patient with poor arterial circulation and small ulcers with sinus tracts on the toes. There were no inflammation signs but drops of light greyish purulent exudate coming from the sinus tracts for many weeks. repeated bacterial analysis showed no growth of bacteria. After a few months we did a fungal test which showed abundant fungal growth.   The patient rinsed the sinus tracts daily with a vinegar-zinc solution and the sinus tracts healed within a few weeks.  Our theory is that previous repeated antibiotic treatments gave rise to an opportunistic fungus that saw his chance of colonizing the sinuses when the bacteria had moved out.  This story is to show that sometimes it is very useful to keep in mind that fungus infections also can give us problems in chronic wounds and ideally we should be able to test for these.

Another advantage of culturing fungal samples is that it is possible to do resistancy tests for antimycotic treatments in the same way that we test bacterial cultures for antibiotic resistance.

 

So how do we go about doing a test to check for fungi?   Fungi are not as easily cultivated as bacteria - at least the process takes longer.  If you have fungal lesions on relatively intact skin you can take skin scrapings and examine these under the microscope.   If you take a sample from a chronic ulcer or  a wound sinus you will most likely not see anything useful with direct microscopy and a fungal culture is indicated.  For this you can use the same type of swab as for bacterial cultures- Amies medium - and send this to a laboratory for culture. 

The most reliable method for testing for fungi is by PCR analysis. PCR stands for polymerase chain reaction.  This is an expensive but ingenious method for detecting even small quantities of fungal ( or other microbial particles).  In most regions in Africa this is only available at large clinics but we expect that more and more private clinics in Africa will invest in this technology making it gradually more available at least in large urban areas. 

The Aga Khan University in Karachi, Pakistan has written a very informative up-to- date manual  for the diagnosis of fungal infections and made this available worldwide at ecommons.aku.de.  Please refer to this manual for more information on this topic- click on the image below to get to a pdf version of the document.

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